RU2757114C1 - METHOD FOR OBTAINING A LINE OF HUMANISED MICE, TRANSGENIC BY hACE2 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology and medicine and discloses an expressed linearised vector consisting of a constitutive CAG-promoter, a STOP cassette terminating transcription from said promoter and flanked by LoxP sites, and an open frame for reading the Homo sapiens hACE2 gene, containing the full-size cDNA of hACE2, the expression whereof is induced tissue-specifically by introduction of a Cre-recombinase characterised by the fact that the cDNA of the hACE2 gene is embedded in the vector for the recognition sites of the restriction endonucleases BshTI – MluI. The invention also discloses a method for creating the described expression vector, a method for obtaining a line of mice transgenic by hACE2 using the described vector, and a transgenic mouse, tissue-specifically expressing the human angiotensin-converting enzyme-2, transformed by said vector. Due to the STOP-cassette flanked by LoxP sites, contained in the vector, the expression of the hACE2 gene can be tissue-specifically activated in the transgenic mice at any time by induction of doxycycline-dependent expression of a Cre-recombinase.

EFFECT: invention allows for creation of lines of genetically modified humanised mice for use as animal models in the research of the mechanisms of pathogenesis of COVID-19 and development of therapeutic approaches.

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Description

Technology area

The invention relates to the fields of molecular biotechnology and medicine and relates to a humanized mouse strain carrying the human hACE2 gene. The invention can be used for testing vaccines against COVID-19, as well as for a detailed study of the mechanisms of penetration of the SARS-CoV-2 virus.

This work was financially supported by the Ministry of Science and Higher Education of the Russian Federation within the framework of Agreement No. 075-15-2019-1661 of October 31, 2019.

State of the art

The COVID-19 pandemic caused by the SARS-CoV-2 virus began in late 2019 and continues to claim lives, causing damage to the global economy unprecedented in decades. Even if the spread of the virus can be stopped by other methods, the risk of a new outbreak will remain until the COVID-19 vaccine is developed.

Vaccine development, however, is complicated by the lack of adequate animal models for testing. Testing on primates is only suitable for the final stages of testing: they require special conditions and are expensive. Conventional mice cannot be used to test vaccines because the virus is unable to infect cells from wild-type mice. To penetrate human cells, SARS-CoV-2 uses surface proteins ACE2, which normally catalyze the hydrolysis of angiotensin II, leading to a decrease in blood pressure. The homology of human and murine ACE2 is insufficient for virus penetration into murine cells. Based on this, it can be assumed that a transgenic mouse expressing human ACE2 will be an adequate model for the early stages of the development of vaccines against COVID-19.

Transgenic mice are known (Linlin Bao, Wei Deng, Baoying Huang, Hong Gao et al, The Pathogenicity of 2019 Novel Coronavirus in hACE2 Transgenic Mice, Nature, 2020 May 7), constitutively expressing hACE2 in all tissues. The SARS-CoV-2 virus has been shown to enter the cells of these mice, and they show symptoms of COVID-19, up to fatal interstitial pneumonia. However, such mice as a model of COVID-19 disease have a significant drawback: the ACE2 protein is not present in all types of cells in humans, and this can largely determine the nature of the course of the disease. In the work, no histological examination of tissues other than the lungs was carried out. Transgenic mice are also known (Paul B. McChau Jr., Lecia Pewe, Christine Wohlford-Lenane et al, Lethal Infection of K18-hACE2 Mice Infected with Severe Acute Respiratory Syndrome Coronavirus, Journal of Virology, 2007 Jan; 81 (2): 813 -21), in which the expression of the hACE2 gene is under the control of the K18 promoter (keratin 18), which is expressed at a high level in the lung, bronchial epithelium, as well as in the liver, pancreas, prostate gland, thyroid gland, and cardiomyocytes. Until now, no work has been published on the study of SARS-CoV-2 infection using this strain of mice, however, a number of its drawbacks were discovered during the study of SARS-CoV infection. First, after infection with the virus, 100% lethality was observed in transgenic animals, and the lifespan depended mainly on the number of embedded copies of the construct and was several days. Secondly, the experimental animals showed the expression of the hACE2 gene and, accordingly, the inflammatory process in the brain, which is atypical for infection in humans. Third, the short time interval between infection and death makes it difficult to study vaccines in this mouse model, since this time may not be enough to activate memory cells.

In addition, the disadvantages of all models with constitutive expression of the hACE2 gene include its expression at the stage of embryonic development, which can lead to unpredictable consequences. The advantage of animals with inducible expression of the KACE2 gene is also their better safety profile compared to animals with constitutive expression: before induction, animals cannot be carriers of the virus and do not require special housing conditions.

Disclosure of the essence of the invention

In the present invention, the inducible expression of the hACE2 gene is provided using the NIF vector. The NIF vector is designed to randomly insert an expression cassette into the genome of an animal. It possesses a number of elements to ensure efficient expression of the transgene. The insulator dimer HS4 ensures the independence of transgene expression from the chromatin state at the insertion site, preventing the spread of repressive chromatin to the inserted construct. The vector also contains effective transcription terminators, the presence of which increases the level of transgene expression, preventing suppression of expression by RNA interference (RU 2525712 C2). However, the most important component of the NIF vector for us is the STOP cassette flanked by LoxP sites located between the constitutive C AG promoter and the open reading frame of the hACE2 gene. The presence of a STOP cassette makes the expression of the transgene impossible. The STOP cassette, however, can be removed due to Cre recombination. The LoxP / Cre recombinase system is derived from the P1 bacteriophage. The DNA sequence located between the two LoxP sites can be deleted when the Cre recombinase protein appears in the cell. There are lines of transgenic mice in whose cells doxycycline-dependent expression of Cre recombinase can occur. Expression of Cre-recombinase can occur both in all tissues of the body and be tissue-specific due to specific promoters. After crossing transgenic mice, transgenesis of which was carried out using the NIF vector, with transgenic mice expressing Cre recombinase, it is possible to activate the expression of the transgene contained in the NIF vector in the whole body or tissue-specific by doxycycline activation of Cre recombinase expression.

The objective of the claimed invention is to create a line of genetically modified mice containing in their genome the hACE2 gene as part of the NIF vector, allowing to activate the expression of the transgene using Cre-recombinase.

The problem is solved by the fact that a new line of genetically modified mice has been obtained containing the NIF-hACE2 construct in the genome, for which:

1. A linearized NIF vector was obtained, characterized by a size of 11000 base pairs, and the ability to activate the expression of a transgene in response to the action of Cre-recombinase, into which a fragment of 2418 base pairs of SEQ ID NO: 1 containing full length hACE2 cDNA.

2. Was developed a method for obtaining a linearized vector NIF, characterized by the following stages:

a) Cloning the open reading frame of the MCE2 gene into the NIF plasmid at the MluI and BshTI restriction sites

b) Obtaining a linearized NIF vector from a circular plasmid by restriction with endonuclease PvuI with removal of a part of the bacterial backbone 1000 base pairs in length.

3. A method of obtaining a line of mice transgenic for hACE2, characterized by the following stages:

a) microinjection of the linearized NIF vector from item 1 into the pronucleus of a fertilized egg of a mouse hybrid F ₁ (CBA × C57BL / 6);

b) identification of viable zygotes;

c) transplantation of surviving zygotes from point (b) to pseudopregnant female recipients with a copulative plug after transplantation with vasectomized males;

d) obtaining newborn mice on day 19 after transplantation of zygotes from point (c);

e) analyzing the presence of the NIF vector according to claim 1 8-14 days after birth of mice (d) using the sequences SEQ ID NO: 2 and SEQ ID NO: 3;

e) selecting mice carrying the NIF vector according to claim 1;

g) crossing mice from point (e) with wild-type mice to obtain heterozygotes carrying the NIF vector containing the transgene.

h) crossing heterozygous mice from point (g) to obtain homozygous mice and the final line of mice transgenic for the hACE2 gene.

EFFECT: developed a method for producing a line of transgenic mice expressing the hACE2 gene after exposure to Cre-recombinase.

The invention is illustrated in the figure and sequence listing.

Brief Description of Drawings

The figure shows a diagram of a NIF vector. In front of the site for cloning the gene of interest, there is a STOP cassette flanked by LoxP sites. Before recombination, the gene is turned off. Tissue-specific activation of Cre-recombinase allows tissue-specific activation of the expression of the target gene.

Implementation of the invention

The method is carried out as follows:

1. Obtaining genetic constructs. cDNA of the hACE2 gene was obtained on the basis of total mRNA obtained from human kidney biopsy material homogenized on a Precellys 24 homogenizer and isolated with the QIAGEN RNeasy Mini Kit .. Reverse transcription was performed using Superscript II reverse transcriptase (Invitrogen) and d (T) i6 primer (SEQ ID NO: 4). Pre-amplification of the hACE2 coding sequence was performed using the primers hACE_5_fwd (SEQ ID NO: 5) and hACE_3_rev (SEQ ID NO: 6) with DNA polymerase Q5 (NEB). The hACE2 gene was amplified using the hACE2-F-BshTI (SEQ ID NO: 7) and hACE2-R-MluI (SEQ ID NO: 8) primers and Phire polymerase (Thermofisher Scientific). The amplified fragment was cut with restriction endonucleases MluI-FD and BshTI-FD (Thermofisher Scientific) and ligated into a vector treated with the same restriction enzymes. The ligation mixture was transformed into competent DH5a cells, colonies were screened, grown in overnight culture, plasmid DNA was isolated from them and checked by restriction analysis. Two samples were sequenced and checked if the sequence was exactly as predicted (SEQ ID NO: 1). One fully tested plasmid was linearized using restriction endonuclease PvuI, purified in agarose gel and isolated using a QIAquick Gel Extraction Kit (Qiagen), and diluted to a microinjection concentration of 1ng / μL.

2. Obtaining mouse eggs. Eggs for microinjection were obtained by the method of induction of superovulation. For this, immature females F _i hybrids (CBA × C57BL / 6) weighing 12-13 g were injected intraperitoneally with 8 units. gonadotropin of the serum of pregnant mares (PMSG) and after 48 hours - 8 units. human chorionic gonadotropin (HCG). After this treatment, the females were bred with male F _i (CBA x C57BL / 6). The fact of mating was ascertained the next morning by the presence of a copulatory plug.

Superovulation induction scheme: 12:00 - PMSG, after 48 hours - HCG. At 17:00 on the same day - replanting to sire males. Donors were selected the next day at 9:00. The light regime in the vivarium was set from 7:00 to 19:00.

The selected donor females were sacrificed, the oviducts were removed, then the oocytes were washed out in HEPES-KSOM medium supplemented with hyaluronidase. The procedure was performed under a binocular microscope (Zeiss Stemi DV4) with a 32-fold magnification. To wash out the eggs, glass capillaries with an inner diameter of about 100 μm were used, made on a Narishige PC-10 puller (Japan) and a Narishige MF-900 micro forge (Japan).

3. Microinjection. The resulting zygotes were cultured for two hours at t = 37 ° C and 5% CO ₂ in a drop of KSOM medium under mineral oil (Sigma, USA), then placed in a microinjection chamber. Microinjections were performed in a HEPES-KSOM medium under a Zeiss Axiovert 200M microscope at a magnification of 400-600 times using Narishige micromanipulators. A Sutter instrument Co P-97 (USA) puller was used for the manufacture of microinjection needles; a Narishige PC-10 puller and a Narishige MF-900 micro forge were used for the manufacture of a holding pipette.

After the end of microinjections, the surviving zygotes were transferred into a drop of KSOM medium under mineral oil (Sigma) and cultured for 1 hour to identify viable embryos.

4. Getting female recipients. Female recipient oocytes prepared as follows: adult female F ₁ (CBA × C57BL / 6) weighing at least 24 g vazektomirovannymi chafing with males of the same line. After 18 hours, pseudopregnant recipients were selected for the presence of copulatory plugs. The zygotes that survived after microinjection were transplanted into the oviducts of a pseudopregnant female. 6-10 embryos were transferred to one pseudopregnant female. The operation was performed under general anesthesia (a mixture of zoletil and rometar was administered intraperitoneally).

5. Operation of vasectomy. The vasectomy operation was performed in advance under general anesthesia (a mixture of zoletil and rometar was injected intraperitoneally). Through an incision in the skin and abdominal wall, the testis, epididymis and vas deferens were pulled out of the abdominal cavity, after which the vas deferens was destroyed with red-hot forceps. The organs were returned to the abdominal cavity, and the entire procedure was repeated on the other vas deferens. At the final stage, sutures were applied to the abdominal wall and the skin, followed by antiseptic treatment of the operating field.

6. Obtaining newborn mice. On day 19 after the transfer of microinjected embryos, the recipient was sacrificed by cervical dislocation and a caesarean section was performed, after which the surviving pups were placed in a pre-prepared nurse. 8-14 days after birth, a tissue sample was taken from the mice, DNA was isolated, and the presence of the transgene was analyzed by PCR.

7. Analysis of the presence of a transgene in the genome of mice by PCR. DNA for PCR was isolated from tissues according to a standard protocol. Amplification of DNA fragments was performed using the GenPak ™ PCR Core kit (Isogene) in the presence of 10 pM hACE-screen-forv (SEQ ID NO: 2) and hACE-screen-rev (SEQ ID NO: 3) primers in the following modes: denaturation 95 ° С - 3 min; then 35 cycles followed: 95 ° С - 40 sec, 58 ° С - 40 sec; 72 ° С - 40 sec; and the last synthesis 72 ° C - 2 min. In addition, PCR was performed with a pair of primers to the STOP cassette (StopC rt F, SEQ ID NO: 9 and StopC rt R, SEQ ID NO: 10) to improve the reliability of the result.

Sequence listing

<110> Federal State Budgetary Institution of Science Institute

gene biology of the Russian Academy of Sciences (Institute of Gene Biology

Russian Academy of Sciences)

<120> Method for obtaining a humanized mouse strain, transgenic for hACE2

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att gag gaa cag gcc aag aca ttt ttg gac aag ttt aac cac gaa gcc gaa gac ctg ttc 120

tat caa agt tca ctt gct tct tgg aat tat aac acc aat att act gaa gag aat gtc caa 180

aac atg aat aat gct ggg gac aaa tgg tct gcc ttt tta aag gaa cag tcc aca ctt gcc 240

caa atg tat cca cta caa gaa att cag aat ctc aca gtc aag ctt cag ctg cag gct ctt 300

cag caa aat ggg tct tca gtg ctc tca gaa gac aag agc aaa cgg ttg aac aca att cta 360

aat aca atg agc acc atc tac agt act gga aaa gtt tgt aac cca gat aat cca caa gaa 420

tgc tta tta ctt gaa cca ggt ttg aat gaa ata atg gca aac agt tta gac tac aat gag 480

agg ctc tgg gct tgg gaa agc tgg aga tct gag gtc ggc aag cag ctg agg cca tta tat 540

gaa gag tat gtg gtc ttg aaa aat gag atg gca aga gca aat cat tat gag gac tat ggg 600

gat tat tgg aga gga gac tat gaa gta aat ggg gta gat ggc tat gac tac agc cgc ggc 660

cag ttg att gaa gat gtg gaa cat acc ttt gaa gag att aaa cca tta tat gaa cat ctt 720

cat gcc tat gtg agg gca aag ttg atg aat gcc tat cct tcc tat atc agt cca att gga 780

tgc ctc cct gct cat ttg ctt ggt gat atg tgg ggt aga ttt tgg aca aat ctg tac tct 840

ttg aca gtt ccc ttt gga cag aaa cca aac ata gat gtt act gat gca atg gtg gac cag 900

gcc tgg gat gca cag aga ata ttc aag gag gcc gag aag ttc ttt gta tct gtt ggt ctt 960

cct aat atg act caa gga ttc tgg gaa aat tcc atg cta acg gac cca gga aat gtt cag 1020

aaa gca gtc tgc cat ccc aca gct tgg gac ctg ggg aag ggc gac ttc agg atc ctt atg 1080

tgc aca aag gtg aca atg gac gac ttc ctg aca gct cat cat gag atg ggg cat atc cag 1140

tat gat atg gca tat gct gca caa cct ttt ctg cta aga aat gga gct aat gaa gga ttc 1200

cat gaa gct gtt ggg gaa atc atg tca ctt tct gca gcc aca cct aag cat tta aaa tcc 1260

att ggt ctt ctg tca ccc gat ttt caa gaa gac aat gaa aca gaa ata aac ttc ctg ctc 1320

aaa caa gca ctc acg att gtt ggg act ctg cca ttt act tac atg tta gag aag tgg agg 1380

tgg atg gtc ttt aaa ggg gaa att ccc aaa gac cag tgg atg aaa aag tgg tgg gag atg 1440

aag cga gag ata gtt ggg gtg gtg gaa cct gtg ccc cat gat gaa aca tac tgt gac ccc 1500

gca tct ctg ttc cat gtt tct aat gat tac tca ttc att cga tat tac aca agg acc ctt 1560

tac caa ttc cag ttt caa gaa gca ctt tgt caa gca gct aaa cat gaa ggc cct ctg cac 1620

aaa tgt gac atc tca aac tct aca gaa gct gga cag aaa ctg ttc aat atg ctg agg ctt 1680

gga aaa tca gaa ccc tgg acc cta gca ttg gaa aat gtt gta gga gca aag aac atg aat 1740

gta agg cca ctg ctc aac tac ttt gag ccc tta ttt acc tgg ctg aaa gac cag aac aag 1800

aat tct ttt gtg gga tgg agt acc gac tgg agt cca tat gca gac caa agc atc aaa gtg 1860

agg ata agc cta aaa tca gct ctt gga gat aaa gca tat gaa tgg aac gac aat gaa atg 1920

tac ctg ttc cga tca tct gtt gca tat gct atg agg cag tac ttt tta aaa gta aaa aat 1980

cag atg att ctt ttt ggg gag gag gat gtg cga gtg gct aat ttg aaa cca aga atc tcc 2040

ttt aat ttc ttt gtc act gca cct aaa aat gtg tct gat atc att cct aga act gaa gtt 2100

gaa aag gcc atc agg atg tcc cgg agc cgt atc aat gat gct ttc cgt ctg aat gac aac 2160

agc cta gag ttt ctg ggg ata cag cca aca ctt gga cct cct aac cag ccc cct gtt tcc 2220

ata tgg ctg att gtt ttt gga gtt gtg atg gga gtg ata gtg gtt ggc att gtc atc ctg 2280

atc ttc act ggg atc aga gat cgg aag aag aaa aat aaa gca aga agt gga gaa aat cct 2340

tat gcc tcc atc gat att agc aaa gga gaa aat aat cca gga ttc caa aac act gat gat 2400

gtt cag acc tcc ttt tag 2418

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ctggacccta gcattggaa 19

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gaaacagggg gctggttagg 20

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<212> DNA

<213> artifficial sequence

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transcriptions

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tttttttttt tttttt 16

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hACE2 gene read

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<212> DNA

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<212> DNA

<213> artifficial sequence

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<212> DNA

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accgttgttt ccaccgaaga 20

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<212> DNA

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Claims (12)

1. Expression linearized vector consisting of a constitutive CAG promoter, a STOP cassette terminating transcription from this promoter and flanked by LoxP sites, and an open reading frame of the Homo sapiens hACE2 gene containing full-length hACE2 cDNA, the expression of which is induced by the introduction of tissue-specific recombinase, characterized in that the cDNA of the hACE2 gene is inserted into the vector at the recognition sites of the restriction endonucleases BshTI - MluI. 2. A method for creating an expression vector according to claim 1, comprising the following steps: i) cloning the open reading frame of the hACE2 gene into a circular plasmid at the MluI and BshTI restriction sites; ii) obtaining a linearized vector from a circular plasmid by restriction with endonuclease PvuI to remove a portion of the bacterial backbone 1000 base pairs in length. 3. A method of obtaining a line of mice transgenic for hACE2 using the expression vector according to claim 1, comprising the following steps: i) introducing the vector from claim 1 into the pronucleus of a fertilized egg of a mouse hybrid F ₁ (CBA * C57BL / 6) and obtaining zygotes; ii) transplanting surviving zygotes into pseudopregnant recipient females having a copulative plug after transplanting with vasectomized males; iii) obtaining newborn mice after zygote transplantation; iv) selecting mice from item iii) carrying the vector according to item 1; v) carrying out the crossing of the resulting mice with wild-type mice to obtain heterozygotes carrying the vector according to claim 1, containing the transgene; vi) carrying out the crossing of heterozygous mice from v) to obtain homozygous mice and the final strain of mice transgenic for the hACE2 gene. 4. Transgenic mouse, tissue-specific expressing human angiotensin-converting enzyme-2, transformed with an expression linearized vector consisting of a CAG promoter, a STOP cassette terminating transcription from this promoter and flanked by LoxP sites, and an open reading frame of the hACE ID NO2 gene with the sequence SEQ 1 containing the full-length cDNA of the hACE2 gene, and the cDNA of the hACE2 gene is inserted into the vector at the recognition sites of the restriction endonucleases BshTI - M1uI.

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